Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the etiologic agent of KS, primary effusion lymphoma (PEL) and multicentric Castleman's disease. These tumors occur in AIDS and other immune compromised states and current therapies are limited. KS is the leading AIDS malignancy, is epidemic in sub Saharan Africa, and often involves the oral cavity and visceral organs. KSHV latently infects tumor cells and viral genomes persists as multiple copy, extrachromosomal, circular episomes. In order to persist in dividing cells, episomes must replicate and efficiently segregate to daughter nuclei. The latency-associated nuclear antigen (LANA) mediates KSHV episome persistence and is essential for latency and virus survival. There are two components to episome persistence: 1) replication of KSHV DNA and 2) segregation of replicated episomes to daughter cell nuclei. To segregate episomes to daughter nuclei, LANA tethers KSHV terminal repeat (TR) DNA to mitotic chromosomes. The tethering occurs through the C- terminal LANA DNA binding domain (DBD) binding TR DNA and N-terminal LANA simultaneously binding histones H2A/H2B on the nucleosome surface. In addition, we recently discovered an internal LANA sequence critical for segregation that does not bind DNA or chromosomes, suggesting it is likely involved in aspects of the segregation process that remain to be elucidated. LANA's ability to segregate episomes is essential to KSHV latency, yet little is known regarding the mechanisms underlying this process. The process of segregation is likely complex, with important questions unanswered. For instance, how does the recently discovered internal LANA sequence contribute to segregation? During mitosis, does random episome attachment occur to chromosomes resulting in random distribution to daughter cells, or is there tight regulatory control targeting each replicated episome to a different sister chromatid for equal distribution to daughter cells? Fundamental questions as to how LANA binds TR DNA to tether episomes to mitotic chromosomes are unanswered. An understanding of these questions is critical to gaining insight into this key aspect of KSHV biology. This proposal investigates the process of LANA mediated segregation of episomes to daughter cells, which is essential for latency and virus survival. Work will define and functionally characterize the recently identified critical internal LANA effector sequence for segregation. State of the art live cell microscopy will be used to investigate episome replication and segregation dynamics. Work will also solve the structure of LANA complexed with DNA, which is central to an understanding of LANA's tethering mechanism. This work will provide comprehensive insight into LANA's essential segregation function and potentially lead to strategies that prevent or treat KSHV associated malignancies.